Genome-Scale Perturbation of Long Noncoding RNA Expression Using CRISPR Interference.
Genome-Scale Perturbation of Long Noncoding RNA Expression Using CRISPR Interference.
复制标题
DOI：
10.1007/978-1-0716-1158-6_20
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Lim DA
中科院分区：
文献类型：
--
作者：
Liu SJ;Horlbeck MA;Weissman JS;Lim DA
CRISPR介导的干扰（CRISPRi）是一种用于可编程地抑制转录的稳健且特异性的系统，为系统地表征长非编码RNA（lncRNA）的功能提供了通用工具。当与高度平行的慢病毒合并筛选方法一起使用时，CRISPRi能够在单次筛选中靶向敲除数万个表达lncRNA的基因座。在这里，我们描述了CRISPRi在合并筛选中靶向lncRNA基因座的用途，使用细胞生长和增殖作为表型读数的示例。还讨论了定制lncRNA靶向文库、替代表型读数和正交验证方法的考虑因素。
CRISPR-mediated interference (CRISPRi), a robust and specific system for programmably repressing transcription, provides a versatile tool for systematically characterizing the function of long non-coding RNAs (lncRNAs). When used with highly parallel, lentiviral pooled screening approaches, CRISPRi enables the targeted knockdown of tens of thousands of lncRNA-expressing loci in a single screen. Here we describe the use of CRISPRi to target lncRNA loci in a pooled screen, using cell growth and proliferation as an example of a phenotypic readout. Considerations for custom lncRNA-targeting libraries, alternative phenotypic readouts, and orthogonal validation approaches are also discussed.